1. Field of the Invention
The present invention relates to antibodies specific for epitopes of human androgen receptor in which a serine residue is phosphorylated. The present invention also relates to a method for determining the presence of activated androgen receptors and to methods for screening an androgen agonist or antagonist/inhibitor.
2. Description of the Related Art
The androgen receptor (AR) is a member of the steroid receptor (SR) family of transcriptional regulatory proteins that transduces the signaling information conveyed by androgens (Chang et al., 1995 and Wilson et al., 1991). Androgen steroid hormones direct the genetic program dictating male development. Upon androgen binding, the androgen receptor is released from the repressive effects of an Hsp90-based regulatory complex, allowing the receptor to either activate or inhibit transcription of target genes in a hormone-dependent manner (Suina et al., 1996; Fang et al., 1996; Fang et al., 1998; Picard et al., 1990; Segnitz et al., 1997; Jenster et al., 1991; and Jenster et al., 1992). In addition to the role the androgen receptor plays in male sex determination, activation of the receptor also mediates normal prostate development and malignant growth by regulating genes involved in cellular proliferation (Brinkmann et al., 1992; Dorkin et al., 1997; Hakimi et al., 1996; Trapman et al., 1996 and Jenster et al., 1999). For example, activation of the androgen receptor is not only responsible for male sexual development, it also plays a critical role in the development and progression of benign prostate hyperplasia, prostate cancer, and hair loss. The androgen receptor controls gene expression through binding with critical transcriptional regulatory proteins (coactivators and corepressors) that, in turn, allow the androgen receptor to “switch on” or “switch off” genes important for malignant prostate cell growth, benign prostate hyperplasia, and androgen-dependent hair loss.
The mechanisms underlying the specificity of AR regulation of gene expression remain enigmatic, however. Functional mapping of the androgen receptor shows that several protein regions are required for transcriptional activation (Jenster et al., 1995 and Chamberlain et al., 1996). These regions include a carboxyl-terminal domain called AF-2, as well as two regions which map to the N-terminus called AF-1a and AF-1b (FIG. 1). Recent evidence suggests that the AR cell- and promoter-specific transcriptional response is generated through interactions with regulatory proteins termed coactivators and corepressors with AF-1 and AF-2 (Cleujens et al., 1997 and Scheller et al., 1998; Chamberlain et al., 1996; Hsiao et al., 1999; Kang et al., 1999; Moilanen et al., 1998; Muller et al., 2000; Aarnisalo et al., 1998; Fronsdal et al., 1998; Fujimoto et al., 1999 and Heinlein et al., 1999).
Agonist binding to the AR C-terminal ligand binding domain promotes a conformational change and the formation of a surface for protein-protein contacts between AF-2 and additional transcriptional regulatory factors, which in turn, modulate the transcriptional activity of target genes (Glass et al., 2000 and Westin et al., 2000). Like other steroid hormone receptors, the AR is phosphorylated upon ligand binding, although the function of this phosphorylation is not well understood. The identified sites of phosphorylation within the AR include serines 16, 81, 94, 256, 308, 424, 650 (Zhou et al., 1995; Zhu et al., 2001 and Gioeli et al., 2002). Recent reports indicate that AR and the serine/threonine cytoplasmic kinase, Akt, physically interact, resulting in effects on AR-mediated transcription, AR stability and cell survival. Sequence analysis of AR indicates that it possesses two consensus Akt phosphorylation sites RXRXXS/T (SEQ ID NO:3), one located within the amino terminal domain and one within the carboxyl terminal domain. The residues, serine 213 and serine 791 have been identified as sites phosphorylated by kinase, Akt (Wen et al., 2000; Lin et al., 2001 and Lin et al., 2002).
At present, androgen receptor activity can only be altered by removing the hormone, testosterone, or by surgical or pharmacological means. Unfortunately, this approach is often short-lived, with androgen-expressing cells learning to grow in the absence of testosterone. Once this has occurred, there is no effective treatment for androgen-dependent afflictions.
Although it is well established that the AR plays a fundamental role in prostate cancer, detection of total AR levels in individual patient samples has not been predictive in determining which patients are at risk for rapid disease progression. While commercially available AR antibodies have allowed investigators to examine AR protein expression in the prostate, it is not clear whether or not the detected androgen receptor is active/ligand bound.
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